Isolierung und Kultur von adulten neuralen Stammzellen aus der Maus subcallosa Zone
Summary
Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.
Abstract
Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.
Introduction
NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro.
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.
Protocol
Representative Results
Discussion
Dieses Dokument beschreibt ein detailliertes Protokoll NSC von der adulten Maus SCZ zu erzeugen und sie für verschiedene Anwendungen zu erhalten. Es gibt drei wichtige Schritte zur Ermittlung der In – vitro – Kultursystem erforderlich zu reinigen und SCZ-NSCs erweitern. Erstens ist es wichtig, sicherzustellen , dass die SCZ Region genau von anderen potentiellen neurogener Bereiche (1B) präpariert wird. Dick und präzise Abschnitte der SCZ Regionen enthielten , wurden mit einem 1 mm A…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C3347).
Materials
| Medium components | |||
| DMEM/F12 | Gibco | 11320-033 | +L-glutamin, +Sodium bicarbonate |
| Pen/Strep | Invitrogen | 15140-122 | |
| N2 supplement | Gibco | 17502-048 | |
| B27 supplement | Gibco | 17504-044 | |
| Growth factor | |||
| bFGF | R&D | 233-FB | |
| EGF | Gibco | PHG0313 | |
| Buffer | |||
| PBS (10X) | BIOSOLUTION | BP007a | 1X dilution |
| HBSS | Gibco | 14175-095 | |
| Dissociattion buffer | |||
| Dispase II | Roche | 04-942-078-001 | |
| Papain | Worthington | 3126 | |
| Accutase | ICT | AT-104 | |
| Tool | |||
| Fine forceps | WPI | 555229F | |
| Scissors | Storz | E3321-C | |
| Brain matrix (1mm) | RWD | 68707 | |
| Double-edged razor | DORCO | ST-300 | |
| 30 gage needle | SUNGSHIM | N1300 | |
| Materials | |||
| 15 ml tubes | SPL | 50015 | |
| 50 ml tubes | SPL | 50050 | |
| 35mm dish | SPL | 10035 | petridish |
| 100mm dish | SPL | 10090 | petridish |
| Cover slip (18mm) | Deckglaser | 111580 | |
| 12 well dish | SPL | 32012 | non-coating |
| 6 well dish | SPL | 32006 | non-coating |
| Coating materials | |||
| PLO | Sigma | P4957 | 0.01% |
| Laminin | Gibco | 23017-015 | 10 mg/ml |
| Primary antibodies | |||
| Nestin | Millipore | MAB353 | mouse (1:1000) |
| EGFR | Abcam | ab2430 | rabbit (1:1000) |
| DCX | Santa Cruz | SC8066 | goat (1:500) |
| Tuj1 | Sigma | T2200 | rabbit (1:2000) |
| GFAP | Invitrogen | 13-0300 | rat (1:1000) |
| O4 | Millipore | MAB345 | mouse (1:500) |
| BrdU | Abcam | ab6326 | Rat (1:500) |
| Secondary antibodies | |||
| anti-mouse 488 | Invitrogen | A21202 | 1:500 |
| anti-mouse cy3 | Jackson | 715-165-151 | |
| anti-mouse 647 | Jackson | 715-606-150 | |
| anti-rabbit 488 | Alexa | A21206 | |
| anti-rabbit cy3 | Jackson | 711-165-152 | |
| anti-rabbit 647 | Jackson | 711-605-152 | |
| anti-goat 488 | Alexa | A11055 | |
| anti-goat cy3 | Jackson | 705-165-147 | |
| anti-goat 647 | Invitrogen | A21447 | |
| anti-rat 488 | Invitrogen | A21208 | |
| anti-rat cy3 | Jackson | 712-166-150 | |
| anti-rat 647 | Jackson | 712-605-153 | |
| Immunostaining materials | |||
| BSA | Millipore | 82-100-6 | 3% BSA with 0.1% Triton X-100 in PBS |
| Triton X-100 | usb | 22686 | |
| 4% PFA | Biosesang | P2031 | |
| Hoechest33342 | Life Technology | H3570 | Dye for staining nuclei |
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